KR100444809B1 - Low pathogenic live vaccine and its preparation method - Google Patents

Abstract

The live or modified live PRRS vaccine of the present invention for administration to pigs is low in toxicity and provides effective immunity against PRRS. More preferred vaccines contain virus isolates having an average plaque diameter of less than about 2 mm and having low pathogenicity. A preferred vaccine is a strain having a small plaque diameter, ATCC Accession No. Contains VR 2509. The vaccines of the present invention can be administered to raised sows, young sows and weaned pigs and are effective in immunizing pigs against diseases of the respiratory and reproductive disorders.

Description

Low pathogenic live vaccine and its preparation method

BACKGROUND OF THE INVENTION [

Over the past few years, PRRS has emerged as a major viral disease of pigs. PRRS causes severe reproductive failure in the pregnant sows, causing signs of preterm birth, increasing the number of stills, reducing the number of litters, and delaying recovery to the estrus. In general, the severe reproductive syndrome of PRRS lasts for two to four months at the farm, but respiratory illness can last for years, resulting in serious loss of productivity. Several studies have been reported on the virulence of PRRS virus infections in the late pregnancy of pregnant sows (77-95 days of gestation). Each study describes the nature of the PRRS virus that causes light placenta infection and fetal virulence. However, there is no clear explanation for the fetal virulence of sows in the mid-trimester period, and the fetus in mid-gestation infected in the uterus is present in a greatly erroneous state.

Depending on farmland conditions, there are farms that are seropositive to PRRS but not severe genital or chronic respiratory disease. In these cases, they are not well aware of the basics without clinical signs of disease. There is a low pathogenic PRRS strain for this phenomenon and it is proposed that this causes the infection of sows without clinical signs of PRRS. Several other investigators have published a number of PRRS virus isolates. All these viral isolates are shown to have RNA and lipid-containing envelopes, but are not capable of hemagglutinating the red blood cells of different animal species.

SUMMARY OF THE INVENTION [

The present invention overcomes the above-mentioned problems and provides an improved or modified PRRS vaccine for administration to the sows. The vaccine of the present invention contains an amount of live or transformed live bacteria sufficient to provide an effective immunity to the sows against an inductive wild type PRRS infection. As used herein, " effective immunity " refers to the efficacy of a vaccine that is able to prevent PRRS infection, which is indicative of clinical signs of disease in the sows. In other words, the immunized sows may or may not be seropositive for PRRS, but no clinical signs at all.

The vaccine of the preferred form of the present invention is a novel live strain named MN-Hs, deposited at the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, MD 20852, 1995. VR 2509. This virus is known to be practically non-toxic and provides effective immunity. The vaccine may be administered to raise sows, young sows, fowls or weaned young pigs, and such administration may be by any conventional method, such as intramuscular injection or oral-nose injection. Generally, the dose of the vaccine contains a unit of virus that forms about 10 ⁴ to about 10 ⁸ plaques.

In general, the present invention also relates to a method for producing a PRRS virus strain or isolate having an average plaque diameter of about 2 mm or less, using a confluentron of MARC-145 cells, by plaque cloning method, Wherein the method comprises the steps of: The MARC-145 cell line is deposited with the ATCC, It is given in CRL 12219. The vaccine of the present invention is preferably composed of such a small plaque diameter virus obtained in a substantially purified form. Virus ATCC Accession No. VR 2509 has this small plaque diameter and is non-toxic and provides immunity as described above.

The present invention relates to a low pathogenic live vaccine administered to a pig to provide an effective immunity against the pig against reproductive and respiratory syndrome (PRRS) virus infection of the pig. In particular, the present invention relates to methods for immunizing pigs against such live bacteria and PRRS viruses and methods for producing such vaccines. In addition, the novel isolated purified low pathogenic PRRS virus, ATCC Fluid Session No. VR 2509 also forms part of the present invention.

Figure 1 is a photograph showing the size and plaque morphology of the virus isolate MN-Hs (< 2 mm) on the MARC-145 cell line.

Figure 2 is a photograph showing the size and plaque shape of the virus isolate MN-Hs (3-5 mm) on the MARC-145 cell line.

Figure 3 is a photograph showing the size and plaque morphology of the virus isolate MN-Hs (2-3 mm) on the MARC-145 cell line.

The following examples illustrate preferred methods for isolating, identifying, and cloning the low pathogenic PRRS virus strains and preferred methods for producing the vaccine from the strains. Such information is for the purpose of illustration only and is not intended to limit the overall scope of the invention. References published herein are incorporated by reference.

(Example 1)

summary

In this example, the pathogenicity of the small plaque mutant (MN-Hs) of porcine reproductive and respiratory distress syndrome (PRRS) virus was investigated in pregnant sows. The MN-Hs strain was cloned from MN-H virus, a mixture of small plaque (MN-Hs) virus and large plaque (MN-HL) virus. In the first experiment to compare the fetal pathogenicity, MN-Hs, MN-HL, field isolate (MN-W) and cell culture medium (control) were collected for two pregnant sows at 86 days of gestation respectively Inoculated with the cox. All sows except control sows were given births on the date of birth. Infected sows had viremia (fertility) at 7 days after inoculation (PI) and seroconversion at PI 14 days by indirect fluorescent antibody (IFA) test. The two sows infected with the MN-Hs virus gave birth to 14 live piglets and 5 dead piglets. The two sows infected with the MN-HL virus did not produce live piglets, He gave birth to a pig. Two sows inoculated with MN-W gave birth to 10 live piglets and 20 dead piglets. Two control sows had 16 normal piglets when slaughtered at 107 days of gestation. Sixteen out of 24 pigs (66.7%), six out of fourteen pigs (64.3%) and three out of 25 dry pigtail pigs (12.0%) out of 24 infected pigs with six infected sows Respectively. 4 Of the 13 sera obtained from infected sows of MN-H _L or MN-W infected sows, six of the 13 sera were in the range of 1:16 to 1: 1,024 of the PRRS virus antibody titer by the IFA method. In a subsequent experiment to repeat the results of the MN-Hs virus, two pregnant sows, each of which had been pregnant for 86 days, were infected with MN-Hs as a co-current, MN-Hs into the muscle as different field isolates (OVL-173 ), Respectively. All sows were given birth on their due dates. Two sows inoculated into the cox and muscle with the MN-Hs virus each produced 15 female piglets, 6 dead piglets, 25 female piglets and 5 dead piglets, while OVL- Two young piglets inoculated with 173 gave birth to six live piglets and 24 dead piglets. These results suggest that the virulence of PRRS virus to sow fetuses differs depending on the virus isolate, and that the MN-Hs strain of PRRS virus is a weak pathogenic virus. The detection of PRRS virus antibodies in the serum of stillborn piglets is useful as a diagnostic method for fetal infections.

Materials and methods

Virus and cell culture

Three different PRRS virus isolates were used in this example. The MN-H isolate was derived from the serum of healthy pigs raised on farms with potential PRRS indications. The MN-H virus is a mixture of viruses with various plaque sizes. To the small plaque (MN-Hs) and a large plaque (MN-H _L) virus from the MN-H virus separated apart, four times plaque purified for each virus for the first experiment and further purified six times for the subsequent experiment Respectively. This is described in Him et al., Am. J. Vet. Res., 52: 1649-1652 (1991). In each plaque process, a single layer of confluent MARC-145 (replication-competent clone (MA-104)) derived from the Green Monkey kidney cell line of Africa was propagated in a 16 mm x 15 mm petri dish, (KIM et al., Arch. Virol., 133: 477-483 (1993)) supplemented with 3% fetal calf serum (FCS), 0.15% sodium carbonate and antibiotics, The culture was inoculated with each virus for 60 minutes at 37 DEG C. After the inoculum was transferred, the culture was washed once with MEM. Then 50 mu g of diethylaminoethyl (DEAE) -dextran / (Disco Laboratories) supplemented with 1.6% of the liquid medium (Disco Laboratories) supplemented with 5 ml of the liquid medium and 5 ml of the liquid medium was added to the dish consisting of the same volume of 2X MEM. Each plate was further incubated at 37 캜 for 5 days in a carbon dioxide incubator. At the end of incubation, The clarified plaques were cloned by picking with a sterile Pasteur pipette and then inoculated on a single layer of uninfected MARC-145 cells. The agar was carefully removed for phosphor coloration and the cell monolayer was then stained for 10 minutes with 2 ml of 1% crystal violet in 20% ethanol. The plates were rinsed with tap water to facilitate irradiation of the plaques.

PRRS virus MN-W was isolated from the farm's diseased pig serum showing typical acute PRRS indications and OVL-173 was obtained from Oxford Veterinary Laboratories, Worthington, MN.

Animal and experimental purpose

Sows about 80 days of gestation were purchased from farms without clinical or serological evidence of infection with PRRS virus. The pigs were fed the pig parvovirus (PPV) and leptospira SPP. Lt; / RTI &gt; twice a year. The exact date of delivery of each sow was obtained. After obtaining each sow, they were housed separately in an isolated room at the University of Minnesota. With two sows _{MN-Hs, MN-H L} , and MN-W virus _{^{(2㎖, 10 5.0~5.5 TClD 50 /}} ㎖) for each pregnancy 86 days was inoculated with kosok. The remaining 2 sows were inoculated with cell culture medium and taken as control. Serum samples from each sow were collected at regular intervals for virus isolation and serological tests. Six infected sows were allowed to nurse naturally, and 2 control sows were slaughtered at 107 days of gestation for fetal survey. Blood and lung samples of dry piglets and dead piglets and dry thoracic fetal chest body fluids were collected for delivery, virus isolation and serum reactions. In the second experiment, MN-Hs virus was further purified six times before inoculation with plaque. Two sows of each pregnancy day 86 were inoculated into the nose of the MN-Hs, into the muscle of the MN-Hs, the cox of various other field isolates (OVL-173), and all the sows I had a baby on the due date. (Marrable et al., A. Agric. Sci., 69: 443-447 (1967)) to determine the length of the crown-rump of the dry gigantized or stillborn fetus in order to estimate the time of death at delivery. . Blood and lung samples were collected and analyzed as described in the first experiment.

Virus isolation and serum test

For virus isolation, the supernatant of each serum sample or lung homogenate was placed in 24-well plate wells and MARC-145 cells (1-2 x 10 ⁵ cells / ml) suspended in MEM supplemented with 3% FCS . The cultures were observed for a typical cytopathic effect (CPE) of PRRS virus for 5-7 days. After the plate was frozen and thawed twice, the supernatant was inoculated into 96-well microplate wells containing freshly marinated MARC-145 cell suspension and incubated for 3-4 days. Evidence of viral infection was investigated by observing CPE and specific fluorescence using a PRRS virus reference that was positive for sera from pigs. Serum from cattle and pigs was tested for antibodies by direct fluorescent antibody (IFA) method (Yoon et al., J. Vet. Diag. Invest., 4: 144-147 (1992)). 96-well test plates were prepared using MARC-145 line cells. Several sera were examined for antibody to PPV by hemagglutination inhibition (H _L ) test as described above (Joo et al., Aust. Vet. J., 52: 422-424 (1976) ).

result

PRRS virus isolates showed different plaque sizes at initial separation. MN-H isolates were cloned into two different populations, MN-Hs and MN-H _L , by plaque size. Each plaque size of MN-Hs and MN-H _L after cloning is less than 2 mm in diameter and in the range of 3 to 5 mm, and in the case of MN-W is 2 to 3 mm (see the drawing).

No significant clinical signs other than weak anorexia detected in 112 and 153 pigs for 5 days after inoculation (PI) were observed in pigs infected with the PRRS virus isolate. The virus was isolated from 6 serum samples from 6 sows on the 7th day after PI, and 1 out of 6 sows on the 14th day after PI. As shown in Table 1, high antibody titer was detected in all sows inoculated on day 14 after PI.

[Table 1]

Viral and antibody responses in sows from 86 days of experimentally infected pregnancy with PRRS virus isolate

[Table 2]

Results of delivery of sows with an 86-day-old pregnancy infected with several different PRRS virus isolates and virus isolates

^a Number of days of pregnancy at the time of delivery or slaughter

^b Crown of dry embryo or stillborn - Lump length (cm)

^{c Number of} isolated sample viruses / number of tested samples

^d slaughtered sows at 107 days of gestation to investigate fetuses

LB - Surviving

SB - Stillbirth

M - dry gangrene

IN - Inside the Nose

IM - Intramuscular

ND - Not Measured

The virus was isolated from one or more embryos of one infant of infected sows. The virus isolate results from each piglet from six infected sows, as in experiment 1, showed that approximately half of the examined fetuses (28 out of 63 pigs) were positive for the virus isolate. Of the 24 pigs tested (66.7%), 9 of the 14 pigs (64.3%) and 3 of the 25 pigs (12.0 %) Showed positive for virus. Ten of the 28 pigs that were positive for the virus from the serum samples were negative for the virus when the lung samples were tested for virus isolates.

153, 147, 68 and 112 were tested for antibody against PRRS virus by IFA and against PPV by H _L , and the results are shown in Table 3. Six of the 13 sera from the dead pigs and two of the 25 thoracic fluids from the dry gangrene pigs had PRRS virus antibodies. IFA was 1: 16 to 1: 1,024, and none of the sera had antibodies against PPV. 17 and 13 of the 14 pork derived from sows 53 is a PRRS virus (IFA titers 1: 64-1: 1,024) and PPV (H _L titers 1: 512-1: 16,384) had antibodies to.

[Table 3]

Detection of antibodies against PRRS virus and PPV in serum obtained from stillborn pigs infected with PRRS virus

^a Crown-lump length (cm)

^b Inverse of IFA or H _L titre

^c LB - survived; SB - stillbirth; M - dry gangrene

Review

This study confirmed the effects of pathogens on the fetus of sows in late pregnancy and the ability of several other PRRS virus isolates to induce a light placenta infection. However, there was a significant difference in virulence between the PRRS virus isolates. Res., 57: 262-268 (1993)), when the multiparous sows were inoculated with the PRRS virus ATCC-VR 2332 on the 93rd day of pregnancy (Christianson, WT et al., Can J. Vet. Res. The birds of the mare and 6.0 fetuses were delivered. In this study, six sows infected with MN-H _L or two field viruses delivered 2.7 live piglets and 11.5 dead piglets on average, and thus these viruses were highly toxic . The pathogenicity between the toxic virus used in this study and ATCC-VR 2332 is different. This is due to the age of the pregnancy at the time of the infection as it is infected with the ATCC-VR virus 7 days later. On the other hand, 6 sows infected with MN-Hs gave birth to an average of 9.0 surviving piglets and 2.7 dead piglets at a time. This result is quite different from the case of toxic virus, suggesting that MN-HS virus is a weak pathogenic strain. It is interesting to note that the main difference between the results of the labor sows infected with MN-Hs and MN-H _L virus virus produced from the same origin observed. Under the same conditions, the MN-Hs and MN-H _L virus gave birth to 5 dead and 25 dead pigs, respectively (P <0.005). From these results, it can be concluded that the virulence of MN-Hs and MN-H _L is significantly different, one being a weak strain and the other having a very high virulence.

Viral isolation from a single piglet infected with the PRRS virus is relatively easy and the virus is isolated at the same rate between the live piglet and the dead piglet. Since the virus can not be recovered from all infected piglets, virus isolation for diagnostic purposes should be attempted from at least two piglets in one go. It has also been found that virus isolation is more conveniently performed from serum than lung tissue samples.

One infant of pigs infected with the toxic virus had an antibody specific for PRRS virus in an amount sufficient to detect one or more stillborn pigs. This finding suggests that antibody detection from stillborn or pre-sacrificed piglets may be a useful method for the diagnosis of abnormal PRRS virus infections in infants. This method is useful for laboratories that have difficulty purchasing virus isolation technology and equipment. Six of the 13 stillborn pigs in the study had positive antibody titer to the PRRS virus and no PPV. This confirms that the detected antibody is from the fetus and is due to the PRRS virus.

It is not known why some of the pigs infected with the PRRS virus do not progress to clinical signs. Experience shows that pigs that are in very good condition before the PRRS virus infection show weaker clinical responses compared to pigs with lower health status. The health status can be explained by the difference of the clinical signs as well as the difference of the strains shown in this study. It is also assumed that the interaction between small plaque viruses and large plaque viruses occurs in host animals and can alter pathogenicity. Even if we have highly virulent PRRS viruses MN-H _L in the farm, MN -H If the virus is isolated, the above assumption will be true if you think that there are few reproductive failure problems on the farm.

(Example 2)

The preferred vaccine according to the invention can be administered to young pigs, sows, fowl or weanling pigs in breeding. Such administration can be administered intramuscularly or into the mouth-nose and can occur at any time. However, vaccination is desirable for young pigs that are just after weaning to protect their later development, growth, and finishing stages to sows that are breeding before breeding to protect their full gestation period.

Generally, the vaccine of the present invention is given in a 2 mL dose containing about 10 ⁴ to 10 ⁸ plaque forming units (PFU), more preferably about 10 ⁶ PFU. Immunization lasts for at least one gestation period in the case of live sows, and vaccination after the weanling of the young piglets will provide immunity for protection during the finish. Preferred vaccines according to the present invention can provide broad cross-protection.

The vaccine according to the invention can induce live bacteria or modified live bacteria (supervised viruses) as well as conventional carriers, bactericides and / or additives.

(Example 3)

summary

In this example, the efficacy of a vaccine consisting of the MN-Hs strain of North America PRRSV to protect 3-week-old pigs from viremia caused by infection with the toxic MN-H _L strain of North America PRRSV was investigated. Respiratory disorders associated with infection by North America PRRSV are primarily due to infection by secondary pathogens present in pig farms. However, clinical signs of respiratory disturbances in pigs infected with North America PRRSV under experimental conditions are not consistently reproduced because secondary pathogens are absent. Therefore, detection of viremia is the best indicator of North America PRRS infection. Absence of viremia, which involves a challenge to the MN-H _L strain makes it suggests that the vaccine injected piglets with MN-Hs strain are that immunization against infection by the MN-H _L strain.

Materials and methods

Three 20-week-old piglets were purchased from PRRS-free farms. Twelve young pigs were vaccinated with co-injected MN-Hs strain (2 ml, 10 ^4.5 TClD50 / ml) and the remaining 8 piglets were taken as control. Vaccinated piglets and control piglets were separately housed in separate rooms. Two weeks after the vaccination, six infected piglets (Pig No 81-86) and four control piglets (Pig No. 93-96) were injected with MN-HL strain (2 ml, 10 ^4.5 TClD ₅₀ / ml) (group I). Six weeks after vaccination, the remaining six vaccinated piglets (piglets No. 87-92) and four control piglets (piglets No. 97-100) were challenged in the same way II). The clinical signs of all piglets were observed daily. (1) Virus isolation using MARC-145 cell culture (Yoon et al., J. Vet. Diag. Invest., 6: 289-292 (1994)) and (2) Serum neutralization Blood samples were collected weekly from each piglet for measurement (Park et al., Am. J. Vet. Res., 52: 1649- 1652 (1991)). The detection of SN antibodies in vaccinated piglets suggests that protective immunity against toxic North American PRRSV occurred.

result

After vaccination, the vaccine virus was isolated from piglets of Groups I and II for up to three weeks after vaccination. No clinical signs were observed in either the vaccinated piglets or control piglets of Groups I and II at challenge time.

Group I

SN antibodies were not detected in any piglets at challenge. After the challenge, the challenge virus was recovered from both vaccinated piglets and control piglets. In addition, viremia progressed in both vaccinated piglets and control piglets (Table 1).

Group II

The vaccinated piglets produced SN antibodies 3 weeks after vaccination and showed a potency of 1: 2 ~ 1: 8 at the challenge. After challenge, the challenge virus was not isolated from the vaccinated piglets, whereas the control piglets underwent viremia (Table 4). These results suggest that vaccinated piglets have acquired protective immunity against toxic North American PRRSV.

[Table 4]

Detection of SN antibodies and viremia in challenged 3-week-old piglets 2 or 6 weeks after vaccination

V = vaccinated, C = control

Challenged with ^a = MN-H _L virus

^b = virus isolation / SN antibody titer

The piglets of Groups I and II were sacrificed at 4 weeks and 8 weeks, respectively, after vaccination.

Claims (13)

A vaccine containing a live virus having the name ATCC VR 2509. The method according to claim 1, Wherein the single dose of the vaccine comprises 10 ⁴ to 10 ⁸ PFU. A method of immunizing a pig against North American swine reproduction and respiratory distress syndrome comprising the step of administering the vaccine of claim 1 to the pig. The method of claim 3, Wherein said pig is a mature breeding stock. The method of claim 3, Wherein the pig is a piglet. The method of claim 3, Wherein said vaccine is administered intramuscularly, by co-current or by mouth. (A) obtaining a live North American PRRSV strain by plaque cloning; (B) preparing a vaccine from the strain obtained in the step (A) Wherein the strain provides a plaque having an average diameter of less than 2 mm when the plaque is produced by a method comprising the steps of: (a) an amount of cells inoculated with said strain, said cell having the designation CRL 12219 (MARC-145), said amount of cells comprising Eagles supplemented with calf serum and 0.15% sodium carbonate in a 3% Min from the confluent monolayer of cells maintained in the minimal basal batch, and the amount is 1 to 2 x 10 &lt; ⁵ &gt; cells per ml of Eagles minimal basal medium supplemented with calf serum and 0.15% sodium carbonate at 3% Containing step, (b) culturing the above-mentioned amount of the cells at 37 DEG C for 60 minutes, (c) transferring the inoculum from the volume of cells, (d) washing said volume of cells with an Eagles minimal basal medium. (e) resuspending the aliquot of cells in 1.6% boiled Noble agar supplemented with 5 ml of 2X Eagles minimal basal medium and 250 占 퐂 diethylaminoethyl (DEAE) -dextran, (f) plating said quantity of cells, (g) providing the plaques by further culturing the aliquots of the cells in a CO ₂ incubator for 5 days at 37 &lt; 0 &gt; C. 8. The method of claim 7, Thereby obtaining the strain in purified form. 8. The method of claim 7, Wherein said strain has the designation ATCC VR 2509. A vaccine comprising a live North American PRRSV strain, wherein said strain provides a plaque having an average diameter of less than 2 mm when the plaque is produced by a method comprising the steps of: (a) an amount of cells inoculated with said strain, said cell having the designation CRL 12219 (MARC-145), said amount of cells comprising Eagles supplemented with calf serum and 0.15% sodium carbonate in a 3% Min from the confluent monolayer of cells maintained in the basal medium, and the amount is 1 to 2 x 10 ⁵ cells per 1 ml of Eagles minimal basal medium supplemented with 3% fetal calf serum and 0.15% sodium carbonate Containing step, (b) culturing the above-mentioned amount of the cells at 37 DEG C for 60 minutes, (c) transferring the inoculum from the volume of cells, (d) washing said volume of cells with an Eagles minimal basal medium, (e) resuspending the aliquot of cells in 1.6% boiled Noble agar supplemented with 5 ml of 2X Eagles minimal basal medium and 250 占 퐂 diethylaminoethyl (DEAE) -dextran, (f) plating said quantity of cells, (g) providing the plaques by further culturing the aliquots of the cells in a CO ₂ incubator for 5 days at 37 &lt; 0 &gt; C. 11. The method of claim 10, Wherein said strain has the name ATCC VR 2509. A strain of live North American PRRSV isolated and purified, wherein said strain has the designation ATCC VR 2509. Wherein the strain provides a plaque having an average diameter of less than 2 mm when the plaque is produced by a method comprising the steps of: isolating and purifying a live North American PRRSV strain, (a) an amount of cells inoculated with said strain, said cell having the designation CRL 12219 (MARC-145), said amount of cells comprising Eagles supplemented with calf serum and 0.15% sodium carbonate in a 3% Min from the confluent monolayer of cells maintained in the basal medium, and the amount is 1 to 2 x 10 ⁵ cells per 1 ml of Eagles minimal basal medium supplemented with 3% fetal calf serum and 0.15% sodium carbonate Containing step, (b) culturing the above-mentioned amount of the cells at 37 DEG C for 60 minutes, (c) transferring the inoculum from the volume of cells, (d) washing said volume of cells with an Eagles minimal basal medium. (e) resuspending the aliquot of cells in 1.6% boiled Noble agar supplemented with 5 ml of 2X Eagles minimal basal medium and 250 占 퐂 diethylaminoethyl (DEAE) -dextran, (f) plating said quantity of cells, (g) providing the plaques by further culturing the aliquots of the cells in a CO ₂ incubator for 5 days at 37 &lt; 0 &gt; C.